For many years it has been recognized that adenosine is a potent and efficacious inhibitor of lipolysis and adenylate and adenylate cyclase activity in white adipose tissue. A1-adenosine receptors in white adipose tissue are tonically active and treatment with antagonists of the A1- adenosine receptors in adipocytes are spontaneously active and cause inhibition of lipolysis even in the absence of an agonist, (2) there is a high receptor reserve for the anti-lipolytic action of adenosine, such that activation of a small fraction of receptors is sufficient to cause a functional response, and (3) there may exist an intracellular pool of A1- adenosine receptors in equilibrium with a concentration of adenosine which exceeds that in the extracellular space. Recent studies in our laboratory have demonstrated the presence of agonist-independent activity of A1-adenosine receptors both in transfected Chinese hamster ovary cells and in membranes prepared from rat epididymal adipose tissue. Adipocytes have a high density of A1-adenosine receptors, a finding consistent with both the presence of spontaneous receptor activity and a high receptor reserve. Membrane caveolae are numerous in adipocytes. Literature reports suggest that caveolae may be a site of adenosine receptor signaling. Therefore the specific aims of the research are (1) determine whether A1-adenosine receptors in the intact adipocyte are active in the absence of an agonist, (2) determine the receptor reserves for adenosine to inhibit accumulation of cyclic AMP and to reduce lipolysis in adipocytes, (3) test the hypothesis that an "intracellular" pool of A1-adenosine receptors mediates a tonic effect of adenosine to inhibit the accumulation of cAMP. Rat isolated epididymal adipocytes will be used for these studies. Agonist independent activity of A1-adenosine receptors will be investigated in Aim 1 by use of inverse agonists and neutral antagonists that have been previously characterized in our laboratory. Receptor reserve for adenosine will be measured in Aim 2 by use of FSCPX, an irreversible antagonist of the A1-adenosine receptor that has been characterized in our laboratory. Recently described methods to determine the concentration of adenosine in the receptor compartment and to isolate caveolar membranes for study will be used in Aim 3. The results of the studies will explain the pharmacologic basis of tonic activity of adipocyte A1-adenosine receptors. The research can be used a prelude to investigation of the excessive activity of A1-adenosine receptors associated with obesity, to investigation of events that regulate spontaneous A1-adenosine receptor activity, and to investigation of subcellular sites of adenosine production and signaling (both agonist- dependent and agonist-independent) in adipocytes.